Factor VIII (previously known as antihaemophilic factor) is a glycoprotein found in circulating plasma at levels of ˜200 ng/mL. The naturally occurring mature single chain protein has a heterogeneous molecular weight of between 265 and 270 kDa depending on glycosylation, though a wider range of 170-280 kDa molecular weight proteins are observed in plasma depending on allelic variation, glycosylation, modification, is fragmentation in vivo. A number of recombinant Factor VIII products is available, and also vary considerably in protein structure and molecular weight.
Plasma Factor VIII has the additional complicating factor that it exists in close association with von Willebrand's Factor (vWF), a complex multimeric protein also involved in clotting. vWF itself is of therapeutic utility, either alone or in conjunction with Factor VIII.
Factor VIII participates in a complex clotting cascade culminating in the conversion of vWF/FVIII to fibrin, and the formation of a fibrin clot. Genetic abnormalities in the production of Factor VIII result in the disease Haemophilia A, which manifests itself primarily in repeated bleeding episodes due to reduced or absent clot formation. Haemophilia A and its symptoms (such as uncontrolled bleeding) can be treated with plasma-derived or recombinant Factor VIII. With appropriate dosing, prophylactic use of Factor VIII can markedly reduce the number and severity of bleeding episodes, including those observed during surgery.
Purification of Factor VIII/vWF from plasma or Factor VIII from recombinant sources is a complex process, generally involving a number of chromatographic and/or precipitation and/or viral inactivation steps. Purification from plasma additionally often involves initial cryoprecipitation and reconstitution steps. These complex purification procedures, coupled with inherent protein instability, result in poor yields of purified product. Immunoaffinity chromatography has been described, but aside from its inherent costliness, the chromatography materials used suffer from a lack of stability and cleanability, resulting in very limited re-use. Peptide ligand-based media suffer from similar problems as immunoaffinity media, and have not found widespread acceptance. It would be desirable to obtain a chromatography material capable of isolating Factor VIII or Factor VIII/von Willebrand's Factor from a number of sources in a yield, purity, and cost-effectiveness heretofore not possible.
WO97/10887 discloses triazine-based compounds, useful as affinity adsorbents, of formula I
wherein R1 is H, alkyl, hydroxyalkyl, cyclohexyl, NH2, phenyl, naphthyl, 1-phenylpyrazole, indazole, benzthiazole, benzoxazole or benzimidazole, any of which aromatic groups can be substituted with one or more of alkyl, alkoxy, acyloxy, acylamino, amino, NH2, OH, CO2H, sulphonyl, carbamoyl, sulphamoyl, alkylsulphonyl and halogen;
one X is N and the other is N, C—Cl or C—CN;
Y is O, S or NR2;
Z is O, S or NR3;
R2 and R3 are each H, alkyl, hydroxyalkyl, benzyl or β-phenylethyl;
Q is benzene, naphthalene, benzthiazole, benzoxazole, 1-phenylpyrazole, indazole or benzimidazole;
R4, R5 and R6 are each H, OH, alkyl, alkoxy, amino, NH2, acyloxy, acylamino, CO2H, sulphonic acid, carbamoyl, sulphamoyl, alkylsulphonyl or halogen;
n is 0 to 6;
p is 0 to 20; and
A is a support matrix, optionally linked to the triazine ring by a spacer.
Compounds of formula I are disclosed as having affinity for proteins such as immunoglobulins, insulin, Factor VII or human growth hormone.
Compounds of related structure are disclosed in WO00/67900 and WO03/097112. They have affinity for endotoxins.